The present invention relates to a microinjection process for introducing an injection substance, particularly foreign, genetic material, into procaryotic and eucaryotic cells, as well as cell compartments of the latter (plastids, cell nuclei), as well as to a nanopipette for performing this process.
Intracellular microinjection of fluorescent dyes (Kempers & van Bel 1997), antibodies (Kamei et al, 1996; Oka et al, 1990; Honer et al, 1988), other proteins (Rose et al, 1992; Staiger et al, 1994; Walton et al, 1992) and genetic material (Kost et al, 1995; Nguyen et al, 1996; Heinzel et al, 1997) is a frequently used method, despite several disadvantages associated with sticking or piercing a microelectrode into a cell. The clear disadvantages of the prior art are associated with damage (cytoplasm loss) to the cell through the glass pipette.
To ensure that the cell is not destroyed in an irreparable manner, the plasma membrane surrounding the cell must close round the stuck in tip of the pipette, so as to prevent cell content leakage. This problem frequently arises on sticking micropipettes (tip diameter 0.5 to 1 .mu.m) into small cells (diameter 10 to 20 .mu.m). In addition, many cells have a high internal pressure, i.e. the turgor pressure (all plant cells up to 4 MPa [40 bar], procaryotic cells and some animal cells), which clearly worsens the problems. Following piercing there is a pressure discharge round the pipette and via the pipette tip into the pipette interior. The latter phenomenon is made visible by the so-called backfiring of the electrode content (Van der Schoot, 1989). Drastic ultrastructural changes by the piercing of pipettes (tip diameter 1 .mu.m and greater) could e.g. be revealed in the case of sieve elements (plant tissue) (Knoblauch and Van Bel, 1998). In the case of pipettes with a tip diameter of 0.1 .mu.m, this phenomenon was not observed. Therefore the aim is to make the pipette tip as small as possible, so that the inflow rate of the cell content into the pipette is lower than the water absorptivity of the cell via its own membrane. Thus, the cell can maintain its internal pressure by water absorption, so as to prevent the backfiring phenomenon. In addition, such a pipette (tip diameter approx. 0.1 .mu.m, i.e. 25 to 100 times smaller than in the conventional case) would cause a much smaller hole in the plasma membrane of the cell and therefore the leakage problems around the perforation point would be minimized. A good sealing action is also to be expected with very small cells.
DE-C2-37 38 874 uses a microneedle with a tip diameter of approx. 1 .mu.m, in order to perform a process for producing genetically transformed plant objects by introducing a transforming factor into the recipient or receptor object, namely into a plant protoplast and subsequent selection of cell lines and plants from said object, which have new, hereditary characteristics, macromolecules being used as the transforming factor and the latter is introduced into the recipient object by microinjection, which is characterized in that the transforming factor is either a DNA molecule or an autonomously replicating organelle and the recipient object is either a single cell or one cell in the cell union.
The negative consequence of such a small tip is the very high pressure required for permitting the injection of substances into a cell through the minute opening. Conventional pressure injection equipment is unsuitable for this, because it is not possible to build up the necessary pressure therewith, so as to be able to press sufficient material into the cell in an appropriate injection time (max a few minutes).